Figure 7. VLP-based vaccination confers protection against lethal POWV-LB challenge.

(A) Vaccination strategy and relevant time points for 10- to 14-week-old B6 mice. Mice were vaccinated with 300 μg per mouse of 2% Alhydrogel (Alum only) ± 2 μg POWV-LB VLP i.m. and allowed to develop a memory response for 21 days post vaccination (dpv). At 21 dpv, mice were boosted with identical formulations of Alum ± 2 mg POWV-LB VLP i.m. Three days after boosting, submandibular bleeds were performed to examine the binding and neutralization activity via ELISA and FRNT. Thirty-one days following initial vaccination, mice were challenged s.c. with 10² FFU POWV-LB and monitored for morbidity and mortality. At 3 dpi, submandibular bleeds were performed to examine CD4⁺ and CD8⁺ T cell responses.

(B) Representative graph from two independent experiments with ELISAs examining binding to whole virions using sera collected from mice 24 dpv with Alum ± 2 μg POWV-LB VLP (n = 8 and n = 9, respectively). Binding indicated by absorbance (A450) to POWV-LB.

(C and D) POWV-Spooner (C) and West Nile virus (WNV) (D) is shown.

(E) Representative graph from two independent experiments with FRNTs performed on BHK-21 cells examining neutralization activity against 10² FFU POWV-LB for sera collected 24 dpv (n = 2 and n = 4, respectively).

(F) Representative flow plots showing T cell responses in blood of mice VLP vaccinated (n = 4) or Alum adjuvant only (n = 3)-treated mice 3 days after 10² FFU POWV-LB challenge. T cells were treated ex vivo with either no stimulus (left), 10 μM peptide (middle), or α-CD3 (right) in the presence of BFA for 6 h and stained and analyzed as in Figure 3.

(G and H) Mortality (G) and weight loss (H) of B6 mice vaccinated and boosted with Alum ± 2 μg POWV-LB VLP(n = 8 and n = 9, respectively) and challenged s.c. with 10² FFU POWV-LB and monitored for 30 dpi. Mice vaccinated with POWV-VLP were protected from lethal POWV-LB challenge (p < 0.0001, Mantel-Cox test). All data are reported as mean ± SEM.